Modified human thymic stromal lymphopoietin

ABSTRACT

Modified, furin resistant human TSLP polypeptides and polynucleotides encoding the modified human TSLP polypeptides are provided. Pharmaceutical compositions, B and T cell activation agents, assays and methods of use are also described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application hereby claims the benefit of U.S. provisionalapplication Ser. No. 60/307,345, filed Jul. 23, 2001, the entiredisclosure of which is relied upon and incorporated by reference.

FIELD OF THE INVENTION

The invention generally relates to recombinant protein expression. Morespecifically, the invention relates to modified human thymic stromallymphopoietin (TSLP) polypeptides that are resistant to degradation inmammalian cell culture, polynucleotides sequences encoding modified TSLPpolypeptides, and processes for the production and use of modified TSLP.

BACKGROUND OF THE INVENTION

Thymic stromal lymphopoietin (TSLP) is a growth factor integral to bothB and T cell development and maturation. In particular, murine TSLPsupports B lymphopoieses and is required for B cell proliferation.Murine TSLP is also critical in controlling the rearrangement of the Tcell receptor-gamma (TCRγ) locus, and has a substantial stimulatoryeffect on thymocytes and mature T cells. See, for example, Friend etal., 1994, Exp. Hematol., 22:321-328; Ray et al., 1996, Eur. J.Immunol., 26:10-16; Candeias et al., 1997, Immunology Letters, 57:9-14.

TSLP possess cytokine activity similar to IL-7. For example, TSLP canreplace IL-7 in stimulating B cell proliferation responses (Friend etal., supra). TSLP and IL-7 appear to mediate their lymphopoietic effectsvia distinct mechanisms. For example, IL-7 activates Janus familytyrosine kinases, including JAK1 and JAK3, and modulates the activity ofthe signal transducer and activator of transcription 5 (STAT5) protein.While TSLP modulates the activity of STAT5, it fails to activate anyJanus family tyrosine kinase members (Levin et. al., 1999, J. Immunol.162:677-683). Although TSLP and IL-7 mediate similar effects on targetcells, they also appear to have distinct signaling pathways and likelysome variation in their biologic response.

The known activities of TSLP in modulating the immune system,particularly in stimulating B and T cell proliferation, development, andmaturation, makes this molecule an attractive therapeutic tool. Theability to produce large quantities of the active polypeptide isessential to commercial production of human TSLP. Production ofrecombinant polypeptides in a mammalian cell expression system is mostcommonly used for commercial human therapeutic applications.

Recombinant huTSLP polypeptide has been expressed in a number ofdifferent expression systems, including mammalian cell lines, asdescribed in WO 00/29581. However, production of useful quantities ofactive huTSLP protein in mammalian cells has been difficult. Inparticular, huTSLP expression in mammalian cells often yields a degradedproduct. Alternative polynucleotide molecules and methods to achieveproduction of useful quantities of active huTSLP polypeptide are needed.

SUMMARY OF THE INVENTION

The amino acid sequence of human TSLP was found to contain a uniquesequence of amino acids containing a furin cleavage site. Modificationsof the polypeptide to inactivate the furin cleavage site, according tothe present invention, provides modified protease resistant huTSLPpolypeptides which are more stable when expressed in mammalian cellsystems as compared with the unmodified TSLP polypeptides.

Modified, protease resistant human TSLP polypeptides of the inventioninclude those having one or more amino acid sequence modifications thatalters and inactivates the furin cleavage site RRKRK, as shown in Table1 below, positioned at approximately amino acid residues 127-131 of SEQID NO: 4. Suitable modifications include amino acid substitutions,deletions, additions, or combinations of these, that alter the aminoacid sequence RRKRK to disrupt the furin cleavage site pattern RXXR, inparticular those that disrupt the pattern RX(R/K)R. Also included arepolypeptides which are substantially similar in amino acid sequence tothe modified huTSLP polypeptides, and fragments thereof, that retain atleast one activity of native TSLP and are protease resistant. In oneembodiment, the sequences RKRK or RKRKV have been deleted from the aminoacid sequence of the huTSLP polypeptides.

The invention also provides polynucleotide molecules encoding themodified protease resistant huTSLP polypeptides discussed above.Polynucleotide molecules of the invention include those having anin-frame nucleic acid sequence modification that disrupts or otherwisedeactivates the codons that encode the furin cleavage site RRKRK [SEQ IDNO: 6] positioned at amino acid residues 127-131 of SEQ ID NO: 4.Suitable modifications of the cleavage site includes in-frame nucleicacid substitutions, deletions, additions, or combinations of these, thatalter the nucleic acid sequence that encodes RRKRK to disrupt theencoded furin cleavage site pattern RXXR, particularly RX(R/K)R.Embodiments include, for example, deletion mutants in which thenucleotide sequence AGG AGA AAA AGG AAA [SEQ ID NO: 5] encoding RRKRK,or the nucleotide sequence AGA AAA AGG AAA GTC [SEQ ID NO: 7] encodingan amino acid sequence RKRKV [SEQ ID NO: 8] have been deleted. Alsoincluded are polynucleotide molecules having sequences which aresubstantially similar to polynucleotide molecules encoding the modifiedTSLP polypeptides, and fragments thereof that retain at least oneactivity of native TSLP and are protease resistant.

The invention also provides additional forms of modified huTSLPpolypeptides, including soluble forms and fusion proteins. For example,the fusion proteins of the invention include modified huTSLPpolypeptides fused to heterologous proteins or peptides that confer adesired function, such as to facilitate purification, oligomerization,stability, secretion or identification of the polypeptide. A fusionprotein of the invention can be produced, for example, from anexpression construct containing a polynucleotide molecule encodingmodified protease resistant huTSLP polypeptide in frame with apolynucleotide molecule encoding the heterologous protein.

The invention also provides vectors, plasmids, expression systems, hostcells, and the like, containing a modified protease resistant huTSLPpolynucleotide molecule. Genetic engineering methods for the productionof modified huTSLP polypeptides of the invention include expression ofthe polynucleotide molecules in cell free systems, cellular hosts, intissues, and in animal models, according to known methods.

The invention further includes compositions containing a substantiallypurified modified huTSLP polypeptide of the invention and an acceptablecarrier. Preferred are pharmaceutical compositions adapted foradministration to cells, tissues, or patients, that are useful to inducethe activities of B cells and T cells in therapeutic treatment, forexample, of immune deficiency disorders, viral infections, and bacterialinfections.

These and various other features and advantages of the invention will beapparent from a reading of the following detailed description and areview of the appended claims.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is a polynucleotide sequence encoding a murine TSLP(GenBank AF232937)

SEQ ID NO: 2 is the amino acid sequence of murine TSLP encoded by SEQ IDNO:1.

SEQ ID NO: 3 is a polynucleotide sequence encoding a human TSLP (GenBankAY037115)

SEQ ID NO: 4 is the amino acid sequence of a human TSLP encoded by SEQID NO:3.

SEQ ID NO: 5 is a polynucleotide sequence AGG AGA AAA AGG AAA, encodinga furin cleavage site RRKRK.

SEQ ID NO: 6 is the amino acid sequence RRKRK encoded by SEQ ID NO: 5.

SEQ ID NO: 7 is a polynucleotide sequence AGA AAA AGG AAA GTC, encodingan amino acid sequence RKRKV present in human TSLP but not murine TSLP.

SEQ ID NO: 8 is the amino acid sequence RKRKV encoded by SEQ ID NO: 7.

SEQ ID NO: 9 is a polynucleotide sequence encoding a modified human TSLPhaving one or more in-frame modifications to the sequence AGG AGA AAAAGG AAA that encodes the furin cleavage site RRKRK, resulting indeactivation of the encoded furin cleavage site.

SEQ ID NO: 10 is the amino acid sequence of the modified TSLP encoded bySEQ ID NO: 9.

SEQ ID NO: 11 is a polynucleotide sequence encoding a modified TSLPpolypeptide having codons AGA AAA AGG AAA encoding amino acids RKRKdeleted.

SEQ ID NO: 12 is the amino acid sequence of the modified TSLPpolypeptide encoded by SEQ ID NO: 11.

SEQ ID NO: 13 is a polynucleotide sequence encoding a modified TSLPpolypeptide having codons AGG AGA AAA AGG AAA encoding amino acids RRKRKdeleted.

SEQ ID NO: 14 is the amino acid sequence of the modified TSLPpolypeptide encoded by SEQ ID NO: 13.

SEQ ID NO: 15 is a polynucleotide sequence encoding a modified TSLPpolypeptide having codons AGA AAA AGG AAA GTC encoding amino acids RKRKVdeleted.

SEQ ID NO: 16 is the amino acid sequence of the modified TSLPpolypeptide encoded by SEQ ID NO: 15.

SEQ ID NO: 17 is a modified human TSLP polypeptide.

SEQ ID NO: 18 is a modified human TSLP polypeptide.

SEQ ID NO: 19 is a PCR forward primer.

SEQ ID NO: 20 is a PCR forward primer.

SEQ ID NO: 21 is a PCR reverse primer.

SEQ ID NO: 22 is a PCR reverse primer.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

The following definitions are provided to facilitate understanding ofcertain terms used frequently herein and are not meant to limit thescope of the present disclosure.

“Amino acid” refers to any of the twenty standard α-amino acids as wellas any naturally occurring and synthetic derivatives. Modifications toamino acids or amino acid sequences can occur during natural processessuch as post-translational processing, or can include known chemicalmodifications. Modifications include, but are not limited to:phosphorylation, ubiquitination, acetylation, amidation, glycosylation,covalent attachment of flavin, ADP-ribosylation, cross linking,iodination, methylation, and the like.

As used herein the term “antibody” refers to intact antibodies includingpolyclonal antibodies (see, for example Antibodies: A Laboratory Manual,Harlow and Lane (eds), Cold Spring Harbor Press, (1988)), and monoclonalantibodies (see, for example, U.S. Pat. Nos. RE 32,011, 4,902,614,4,543,439, and 4,411,993, and Monoclonal Antibodies: A New Dimension inBiological Analysis, Plenum Press, Kennett, McKearn and Bechtol (eds.)(1980)). The term “antibody” also refers to a fragment of an antibodysuch as F(ab), F(ab′), F(ab′)₂, Fv, Fc, and single chain antibodieswhich are produced by recombinant DNA techniques or by enzymatic orchemical cleavage of intact antibodies. The term “antibody” also refersto bispecific or bifunctional antibodies, which are an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including fusion of hybridomas or linking of Fab′ fragments.(See Songsivilai et al, Clin. Exp. Immunol. 79:315-321 (1990), Kostelnyet al., J. Immunol. 148:1547-1553 (1992)). As used herein the term“antibody” also refers to chimeric antibodies, that is, antibodieshaving a human constant antibody immunoglobin domain is coupled to oneor more non-human variable antibody immunoglobin domain, or fragmentsthereof (see, for example, U.S. Pat. No. 5,595,898 and U.S. Pat. No.5,693,493). Antibodies also refers to “humanized” antibodies (see, forexample, U.S. Pat. No. 4,816,567 and WO 94/10332), minibodies (WO94/09817), and antibodies produced by transgenic animals, in which atransgenic animal containing a proportion of the human antibodyproducing genes but deficient in the production of endogenous antibodiesare capable of producing human antibodies (see, for example, Mendez etal., Nature Genetics 15:146-156 (1997), and U.S. Pat. No. 6,300,129).The term “antibodies” also includes multimeric antibodies, or a higherorder complex of proteins such as heterdimeric antibodies. “Antibodies”also includes anti-idiotypic antibodies including anti-idiotypicantibodies against an antibody targeted to the tumor antigen gp72; anantibody against the ganglioside GD3; or an antibody against theganglioside GD2.

“Fc” or “Fc polypeptide” refers to polypeptides containing the Fc domainof an antibody. The “Fc domain” refers to the portion of the antibodythat is responsible for binding to antibody receptors on cells. An Fcdomain can contain one, two or all of the following: the constant heavy1 domain (C_(H)1), the constant heavy 2 domain (C_(H)2), the constantheavy 3 domain (C_(H)3), and the hinge region. The Fc domain of thehuman IgG1, for example, contains the C_(H)2 domain, and the C_(H)3domain and hinge region, but not the C_(H)1 domain. Truncated forms ofsuch polypeptides containing the hinge region that promotes dimerizationare also included. See, for example, C. A. Hasemann and J. Donald Capra,Immunoglobins: Structure and Function, in William E. Paul, ed.

“Antisense” refers to polynucleotide sequences that are complementary totarget “sense” polynucleotide sequence.

“Cell targeting moiety” refers to any signal on a polypeptide, eithernaturally occurring or genetically engineered, used to target apolypeptide to a cell, polypeptide, polynucleotide, or innate material.

“Complementary” or “complementarity” refers to the ability of apolynucleotide in a polynucleotide molecule to form a base pair withanother polynucleotide in a second polynucleotide molecule. For example,the sequence A-G-T is complementary to the sequence T-C-A.Complementarity can be partial, in which only some of thepolynucleotides match according to base pairing, or complete, where allthe polynucleotides match according to base pairing.

As used herein, the term “derivative” refers to a modified resistantTSLP polypeptides attached to at least one additional chemical moiety,or to at least one additional polypeptide to form covalent or aggregateconjugate such as glycosyl groups, lipids, phosphate, acetyl groups, orC-terminal or N-terminal fusion proteins and the like.

“Expression” refers to transcription and translation occurring within ahost cell. The level of expression of a DNA molecule in a host cell canbe determined on the basis of either the amount of corresponding mRNAthat is present within the cell or the amount of DNA molecule encodedprotein produced by the host cell (Sambrook et al., 1989, Molecularcloning: A Laboratory Manual, 18.1-18.88).

As used herein, the term “furin cleavage site” refers to an amino acidsequence recognized and cleaved by furin. In human TSLP, for example, afurin cleavage site has been identified within the sequence RRKRK. Ingeneral, the minimal cleavage site for furin is RXXR and morepreferably, RX(R/K)R. (Nakayama 1997, Biochem J 327:625-35). The term“furin” refers to a calcium dependent serine protease is involved in theprocessing of a variety of proteins. Furin is known to cleave variousproproteins, such as growth factor precursors, into biologically activeproteins. Furin mRNA has been detected in all tissues and cell linesexamined, suggesting that its activity is ubiquitous and not focused onany particular target group of proteins. Examples of preproteins cleavedby furin include various growth factors, growth factor receptors, plasmaproteins involved in blood-clotting and complement cascades, matrixmetalloproteinases, viral-envelope glycoproteins, and bacterialexotoxins.

As used herein, the term “modified TSLP polypeptides” or “modifiedhuTSLP polypeptides” is used interchangeably with “furin resistant TSLP”or “protease resistant TSLP” and refers to any huTSLP polypeptide thathas been modified to inactivate the furin cleavage site RXXR, and thatalso retains a TSLP activity, such as stimulation of B or T cellproliferation or development, or binding to TSLP receptors, asdescribed, for example, in WO 00/29581, or in the Examples below. Theterm “modified TSLP polypeptides” also includes variants and fragmentssuch as the extracellular domain, as well as derivatives such as fusionproteins.

“Fusion protein” refers to a protein having a heterologous polypeptideattached via recombinant DNA techniques. The fused heterologouspolypeptide can provide a specific function, for example, to determinethe location of the fusion protein in a cell, enhance the stability ofthe fusion protein, facilitate purification of the fusion protein, ortarget the protein to a desired antigen or cell. Examples of such fusionproteins include polypeptides fused to a portion of an immunoglobulinmolecule, for example, an Fc fragment, polypeptides fused to a histidinetag, a growth factor, and the like, as described, in WO 00/29581.

“Genetically engineered” refers to any recombinant method used to createa eukaryotic host cell that expresses a protein of interest. Methods andvectors for genetically engineering host cells are well known; forexample, various techniques are illustrated in Current Protocols inMolecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988,and quarterly updates). Genetic engineering techniques include, but arenot limited to, expression vectors, targeted homologous recombinationand gene activation (see, for example, U.S. Pat. No. 5,272,071 toChappel) and transactivation by engineered transcription factors (see,for example, Segal et al., 1999, Proc Natl Acad Sci USA 96(6):2758-63).

“Homology” refers to a degree of complementarity betweenpolynucleotides, where the degree of complementarity betweenpolynucleotide molecules has significant effects on the efficiency andstrength of hybridization between the polynucleotide molecules.

“Host cell” or “host cells” refers to cells established in ex vivoculture. It is a characteristic of host cells discussed in the presentdisclosure that they be capable of expressing furin resistant TSLP, asdefined herein. Examples of suitable host cells useful for aspects ofthe present invention include, but are not limited to, mammalian celllines. Specific examples of such cell lines include human embryonickidney cells (293 cells), Chinese hamster ovary (CHO) cells (Puck etal., 1958, Proc. Natl. Acad. Sci. USA 60, 1275-1281), human cervicalcarcinoma cells (HELA) (ATCC CCL 2), human liver cells (Hep G2) (ATCCHB8065), human breast cancer cells (MCF-7) (ATCC HTB22), human coloncarcinoma cells (DLD-1) (ATCC CCL 221), Daudi cells (ATCC CRL-213), COScells, and CV-1 cells.

“Hybridization” refers to the pairing of complementary polynucleotidesduring an annealing period. The strength of hybridization between twopolynucleotide molecules is impacted by the homology between the twomolecules, stringency of the conditions involved, the meltingtemperature of the formed hybrid, and the G:C ratio within thepolynucleotides.

“Inactivated” refers an activity that has been rendered nonfunctional.For example, a furin cleavage site in a polypeptide can be inactivatedby modifying the amino acid sequence. Cleavage of the modifiedpolypeptide in the presence of furin is reduced, and preferably iseliminated, as compared with the wild type polypeptide. Reduced oreliminated cleavage is demonstrated, for example, by a change in thecleavage products as compared to the cleavage products of the wild type.

“Isolated” refers to a polynucleotide or polypeptide that has beenseparated from at least one contaminant (polynucleotide or polypeptide)with which it is normally associated. For example, an isolatedpolynucleotide is in a context or in a form that is different from thatin which it is found in nature.

As used herein, the term “huTSLP polypeptide” refers to a human TSLPpolypeptide having the amino acid sequence set forth in SEQ ID NO: 4, ora variant or fragment of that polypeptide that retains at least oneactivity of a TSLP having SEQ ID NO: 4. A variant is a polypeptide whichhas an amino acid sequence that is substantially similar to the aminoacid sequence of the unaltered protein, or a fragment thereof. For thepurposes of the present invention, “substantially similar” to is atleast about 80% identical to, preferably at least about 90% identicalto, more preferably at least about 95%, more preferably at least about98%, most preferably at least about 99% identical to the amino acidsequence of the unaltered protein, and which retains the activity of theunaltered polypeptide. Amino acid substitutions which are conservativesubstitutions unlikely to affect biological activity are consideredidentical for the purposes of this invention and include the following:Ala for Ser, Val for Ile, Asp for Glu, Thr for Ser, Ala for Gly, Ala forThr, Ser for Asn, Ala for Val, Ser for Gly, Tyr for Phe, Ala for Pro,Lys for Arg, Asp for Asn, Leu for Ile, Leu for Val, Ala for Glu, Asp forGly, and the reverse. (See, for example, Neurath et al., The Proteins,Academic Press, New York (1979)).

The percent identity may be determined by visual inspection andmathematical calculation, or by a comparison of two sequences usingvarious computer programs used by those of skill in the art. Forexample, the percent identity of two sequences can be determined usingthe GAP computer program, based on the algorithm of Smith and Waterman,Adv. Appl. Math. 2:482-489 (1981), (available from the University ofWisconsin Genetics Computer Group (UWGCG), University Research Park,Madison, Wis.). The preferred default parameters for the GAP programinclude: (1) a scoring matrix, blosum62, as described by Henikoff andHenikoff Proc. Natl. Acad. Sci USA 89:10915 (1992)); a gap weight of 12;(3) a gap length weight of 4; and (4) no penalty for end gaps. Otherprograms used by those skilled in the art of sequence comparision mayalso be used.

“Polynucleotide” refers to a sequence of nucleotides. The nucleotidesare either a sequence of polyribonucleotides orpolydeoxyribonucleotides, or a mixture of both. Examples ofpolynucleotides in the context of the present invention include singleand double stranded DNA, single and double stranded RNA, and hybridmolecules that have both mixtures of single and double stranded DNA andRNA. Further, the polynucleotides of the present invention can includeone or more modified nucleotide.

As used herein the term “protein” and “polypeptide” are usedinterchangeably and is considered to be any chain of at least ten aminoacids linked by peptide bonds. Purification of a protein fromcontaminating proteins can be accomplished through any number of knowntechniques, including, ammonium sulfate or ethanol precipitation, anionor cation exchange chromatography, phosphocellulose chromatography,hydrophobic interaction chromatography, affinity chromatography,hydroxylapatite chromatography, and lectin chromatography. Variousprotein purification techniques are illustrated in Current Protocols inMolecular Biology, Ausubel et al., eds. (Wiley & Sons, New York, 1988,and quarterly updates).

“STAT5” refers to a member of the signal transducers and activators oftranscription (STAT) family of transcription factors known to beactivated by one or more JAK kinase, translocate to the nucleus, andparticipate in transcriptional regulation by binding to specific DNAsites. Techniques for determining STAT5 activity include DNA bindingassays, STAT5 dependent reporter assays, ³²P-labeling of STAT5, and thelike, as illustrated in Current Protocols in Molecular Biology, Ausubelet al., eds. (Wiley & Sons, New York, 1988, and quarterly updates).

“Thymic stromal lymphopoietin” (TSLP) refers to a growth factor thatstimulates the process of hematolymphoid development, as described, forexample, in WO 00/29581, and Sims et al., 2000, J. Exp. Med.192:671-680.

“Vector,” “extra-chromosomal vector”, or “expression vector” refers to afirst polynucleotide molecule, usually double-stranded, which can haveinserted into it a second polynucleotide molecule, for example aheterologous polynucleotide, such as a polynucleotide encoding furinresistant human TSLP. A heterologous polynucleotide may or may not benaturally found in the host cell, and can be one or more additional copyof a nucleic acid sequence naturally present in the host genome. Thevector transports the foreign polynucleotide into a suitable host cell.Once in the host cell, the vector can be capable of integrating into thehost cell chromosomes. The vector can also contain the necessaryelements to select cells containing the integrated polynucleotide, aswell as elements to promote transcription of mRNA from the transfectedpolynucleotide. Examples of vectors within the scope of the presentinvention include, but are not limited to, plasmids, bacteriophages,cosmids, retroviruses, and artificial chromosomes.

Unless otherwise stated, the techniques utilized can be found in any ofseveral well-known references, such as: Molecular Cloning: A LaboratoryManual (Sambrook et al. (1989) Molecular cloning: A Laboratory Manual),Gene Expression Technology (Methods in Enzymology, Vol. 185, edited byD. Goeddel (1991) Academic Press, San Diego, Calif.), “Guide to ProteinPurification” in Methods in Enzymology (M. P. Deutshcer, 3d., (1990)Academic Press, Inc.), PCR Protocols: A Guide to Methods andApplications (Innis et al. (1990) Academic Press, San Diego, Calif.),Culture of Animal Cells: A Manual of Basic Technique, 2^(nd) ed. (R. I.Freshney (1987) Liss, Inc., New York, N.Y.), and Gene Transfer andExpression Protocols, pp 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.).

TSLP Polypeptides

Thymic stromal lymphopoietin (TSLP) is a growth factor, a member of thecytokine family of lymphopoietic signaling factors that is integral toboth B and T cell development and maturation. TSLP promotes B celllymphopoiesis to the B220⁺/IgM⁺ immature B cell stage and induces theproliferation of the factor-dependent cell line NAG8/7. TSLP is involvedin controlling the rearrangement of the T cell receptor-gamma (TCRγ)locus, and has a substantial stimulatory effect on thymocytes and matureT cells.

The biological activities of TSLP on B and T cells partially overlapwith the activities of the cytokine, IL-7. For example, both TSLP andIL-7 co-stimulate thymocytes and mature T cells, sustain the NAG8/7 cellline, and support B lymphopoiesis in fetal liver cells. Theseoverlapping functions likely result from the common use by the TSLP andIL-7 receptor complexes of the IL-7R alpha-chain. (Park et al., 2000, J.Experimental Medicine, 192(5):659-669; Levin et al., 1999, J. Immunol.,162(2): 677-83; Isaksen et al., 1999, J. Immunol., 163(11):5971-7).Blockage of the IL-7 receptor will likely block the activities of bothIL-7 and TSLP.

IL-7, together with IL-15, is important for the development and functionof immune cells, B and T cells. IL-7 aids the development of CD4 and CD8T cells as well as for the proliferation and survival of naïve andmemory CD4 T cells. IL-15 is important for the development of naturalkiller (NK) cells, and is involved in the development of memory CD8cells but not memory CD4 cells.

Using an IL-15 knockout mouse model and an antibody directed against theIL-7 receptor alpha chain, it was determined that IL-7 but not IL-15 isrequired for the proliferation of naïve CD8 T cells, including OTI TgTcells and polyclonal CD 44 lo. Acute homeostasis driven proliferation(HDP) of memory CD8 T cells (OTI or polyclonal CD44 hi) is delayed inIL-15 KO mice and by treatment of wild type mice with anti-IL-7 receptormonoclonal antibody. In the absence of IL-15 and inhibition of IL-7receptor function, memory T cell proliferation is almost completelyinhibited. Basal homeostatic proliferation of CD8 memory T cells isblocked in IL-15 KO mice. Treatment with anti-IL-7 receptor monoclonalantibody delayed proliferation in wild type mice. In the absence ofIL-15 and under inhibition of IL-7 receptor function, survival of the Tcells is affected. These results indicate that IL-7 and IL-15 areessential for the proliferation and survival of CD8 memory T cells.Because TSLP and IL-7 share overlapping functional activities on T cellsvia the IL-7 receptor, it is anticipated that TSLP also functions topromote the proliferation and survival of CD8 memory T cells. The memoryT cell data indicates that TSLP is useful for obtaining long termimmunity, and thus can be used as a vaccine adjuvant.

More particularly, TSLP supports the proliferation and differentiationof committed B220⁺ B cell progenitors in vitro (Ray, et al., 1996, Eur.J. Immunol. 1996, 26:10-16). Cells incubated in the presence of eitherIL-7 or TSLP express cell surface markers characteristic of the pro-Bcell stage of B cell differentiation. TSLP can replace IL-7 during thefirst 4-6 days of in vitro culture to support the progression of B cellprogenitors from uncommitted bipotential precursors. TSLP can alsosubstitute for IL-7 in supporting the sustained proliferative responseexhibited by B cell progenitors from CBA/N mice. TSLP supports theexpansion of B220⁺ pre-B cells from either fetal liver or bone marrowfor several days in vitro. TSLP also facilitates proliferation anddifferentiation of pre-B cells isolated from bone marrow up until thestage of becoming mitogen responsive in the presence of the stromal cellline S17.

TSLP facilitates the expansion and differentiation of B cell progenitorsin vitro, and can replace IL-7 in supporting the development of B cellsfrom B220⁺ precursors as well as uncommitted bipotential progenitors invitro. Techniques for stimulating B lineage and T lineage cellproliferation are well known (Ray et al., 1996 supra; Namikawa et al.,1996, Blood 87(5):1881-1890), as are techniques for expandinghematopoietic cells from sources such as umbilical cord blood and bonemarrow (W. Piacibello, et al., 1997, Blood, 89(8):2644-2653). TSLP,alone or in combination with other cytokines, such as IL-7, can be usedto control and amplify pluripotent stem cell renewal and expansion forcord blood or bone marrow transplantation.

Recombinant IL-7 has been used to reconstitute a patient's immune systemfollowing autologous bone marrow transplantation (Abdul-Hai et al.,1996, Experimental Hematology 24:1416-1422). IL-7 induces proliferationand differentiation of pre-B cells and immature thymocytes. TSLP inducessimilar proliferative effects on pre-B cells. Therefore, TSLP, alone orin combination with other cytokines or growth factors such as IL-7, canbe used to stimulate hematopoietic cell proliferation anddifferentiation.

TSLP induces tyrosine phosphorylation of both isoforms of STAT5 (STAT5aand STAT5b), resulting in STAT5-DNA complex formation and transcriptionof the STAT5-responsive gene CIS, a feedback modulator of STAT5 (Levinet al., supra; Isaksen et al., supra). STAT5 has been extensivelystudied in STAT5-deficient mice. One or both forms of STAT5 plays a rolein modulating the immune system, hematopoiesis, sexually dimorphicgrowth, mammary development, hair growth, deposition of adipose tissue,and pregnancy (Davey, et al., 1999, Am. J. Hum. Genet. 65:959-965). Manycases of freshly isolated human lymphoid leukemic cells have been shownto exhibit constitutive activation of STAT5 (Nosaka, et al. 1999, TheEMBO Journal 18(17):4754-4765).

As one example of a STAT5 regulated activity, STAT5a and STAT5b arerequired for normal mammary gland growth and differentiation (Richer etal., 1998, J. Biol. Chem. 273(47):31317-31326). STAT5a-deficient micelack proliferative mammary lobulo-alveolar outgrowth, and the femalesare unable to lactate. STAT5b-deficient female mice have impairedmammary gland development.

TSLP appears to be a central actor in B and T cell development. TSLPproteins are useful in therapies and treatments targeted at stimulatingthe proliferation and maturation of B and/or T cells, for example in thetreatment immune disorders, such as AIDS. Inhibition of TSLP expression,for example by an anti-TSLP antibody, or engagement of the TSLP receptorwith a non-active TSLP fragment or inhibitory analog of TSLP, caninhibit B and T cell development and proliferation, and therapeuticallyuseful, for example, in the treatment of autoimmune disease or inpreventing rejection of organ transplant.

Human TSLP polypeptides are described in WO 00/29581. The amino acidsequence of one preferred embodiment of the full length human TSLP isgiven in SEQ ID NO: 4. Computer analysis predicts that the maturepolypeptide sequence corresponds to amino acids 29 to 159 of SEQ ID NO:4, while the signal peptide is thought to correspond to amino acids 1through 28 of SEQ ID NO: 4, or alternatively amino acids 1 through 34 or1 through 116. The huTSLP polypeptides may be membrane bound or soluble,secreted polypeptides. In one embodiment, the soluble polypeptide mayinclude all or part of the extracellular domain, but lack thetransmembrane region, which would cause retention of the polypeptide ona cell membrane. Human TSLP polypeptides include variants of thepolypeptide encoded by SEQ ID NO:4 having at least 80% identity in aminoacid sequence to SEQ ID NO:4 and retaining at least one TSLP function,as well as fragments thereof retaining a TSLP function.

Protease Resistance

The nucleic acid sequences encoding murine TSLP (GenBank accessionnumber AF232937) and human TSLP (GenBank accession number AY037115) weredisclosed in PCT application WO 00/29581. As described more fully in theExamples below, expression of human TSLP cDNA in mammalian cells oftenyields a degraded product.

In contrast to human TSLP, murine TSLP was not degraded when expressedin mammalian cells. The nucleic acid and amino acid sequences of humanand murine TSLP were compared, and significant differences were found.In particular, the human nucleic acid sequence encodes a unique stretchof amino acids, 127-RRKRV-132, not present in the murine protein.Further analysis suggested that this unique stretch of amino acidscontained a furin cleavage site, 127-RRKRK-131.

As more fully described in the Examples below, human TSLP proteinoverexpressed and isolated from mammalian cell cultures, when analyzed,for example, by electrophoresis, contains a number of polypeptides,shown as numerous bands on a gel. A prominent band in the mixture ofproteins has a molecular weight of approximately 6 kD. The amino acidsequence of the 6 kD fragment corresponded to the C-terminal end ofTSLP, suggesting a cleavage point at the furin cleavage site, RRKRK.This data provides direct evidence that degradation of human TSLPexpressed in mammalian cells resulted from cleavage at the furincleavage site.

The furin cleavage site is located about 8 residues before the start ofa fourth helix of a four-helix bundle in the amino acid sequence ofhuTSLP thought to be required for activity. Truncation of huTSLP at thisfurin cleavage site produces a three-helix bundle cytokine, and alsoremoves the last of the conserved cysteine residues shared between mouseand huTSLP that is thought to be involved in intramolecular disulfidebond formation. Accordingly, cleavage of huTSLP at the furin cleavagesite is thought to remove a portion of the molecule that is required forbiological activity.

In the present invention, a furin cleavage site in huTSLP has beenidentified, and modified to prevent furin cleavage of huTSLP. Accordingto the invention, one or more of the codons encoding the furin cleavagesite, RRKRK, is altered, for example, by site-directed mutagenesis, toprevent recognition of the cleavage site by furin. Preferably, one ormore codons are altered to disrupt the cleavage site. Since the minimalfurin recognition site is RXXR, any modification that disrupts the RXXRpattern in huTSLP is within the scope of the present invention.

Modified Human TSLP Polypeptides

Modified human TSLP polypeptides of the present invention includespolypeptides having the human TSLP amino acid sequence set forth in SEQID NO: 4, modified to deactivate the furin cleavage site RRKRK [SEQ IDNO: 6], as well as variants having an amino acid sequence that issubstantially similar to the amino acid sequence of SEQ ID NO: 4, orfragments thereof, that are both resistant to furin cleavage and retaina functional activity of human TSLP.

For the purposes of the present invention, the term “substantiallysimilar” refers to least about 80% identical to, preferably at leastabout 90% identical to, more preferably at least about 95% identical to,more preferably at least about 98% identical to, most preferably atleast about 99% identical to the amino acid sequence of the unalteredprotein, and which retain at least some degree of at least one activityof the unaltered polypeptide. Amino acid substitutions which areconservative substitutions unlikely to affect biological activity areconsidered identical for the purposes of this invention and include thefollowing: Ala for Ser, Val for Ile, Asp for Glu, Thr for Ser, Ala forGly, Ala for Thr, Ser for Asn, Ala for Val, Ser for Gly, Tyr for Phe,Ala for Pro, Lys for Arg, Asp for Asn, Leu for Ile, Leu for Val, Ala forGlu, Asp for Gly, and the reverse. (See, for example, Neurath et al.,The Proteins, Academic Press, New York (1979)). Further informationregarding phenotypically silent amino acid exchanges can be found inBowie et al., 1999, Science 247:1306-1310).

Modifications suitable for inactivating the furin cleavage site includesamino acid substitutions, deletions, additions, or combinations ofthese, that alter the amino acid sequence RRKRK to disrupt the furincleavage site pattern RXXR, particularly disrupting the patternRX(R/K)R, (wherein R refers to arginine, K refers to lysine, and Xrefers to any amino acid). In one embodiment the sequence RKRK or RKRKVhas been deleted in the modified TSLP polypeptides of the presentinvention.

Preferably, at least two amino acids within the furin cleavage site arealtered to remove dibasic amino acids arginine or lysine that can berecognized by furin. For example, the modification can result insubstitution of one or more dibasic amino acids with one or more neutralamino acid. The dibasic amino acids can also be deleted, or an insertioncan be made within the 127-131 amino acid region of SEQ ID NO: 4 todisrupt the cleavage site.

In one embodiment, the modified TSLP polypeptides of the inventioninclude deletions of one or more, preferably two or more of the aminoacid residues 127-RRKRK-131 of SEQ ID NO: 4 to disrupt the RXXR furincleavage pattern. For example, deletion of one arginine (R) results inthe disrupted sequence RKRK or RRKK; deletion of two arginines resultsin the disrupted sequence KRK or RKK; deletion of three argininesresults in the disrupted sequence KK. Modified TSLP polypeptides alsoinclude deletions of four or all five basic amino acids, for example,deleting RKRK, RRKR, or RRKRK in the amino acid positions 128-RKRK-131or 128-RKRKV-132 of SEQ ID NO: 4.

In an alternative embodiment, the modified human TSLP polypeptides ofthe invention include amino acid substitutions in the human TSLP aminoacid sequence, wherein one or more, and preferably two or more of theamino acid residues 127-RRKRK-131 are substituted with a different aminoacid residue, disrupting the RXXR pattern. Preferably, one or morearginine and/or lysine is substituted with a non-basic, more preferablya neutral amino acid. By way of example, substitution of one arginine(R) results in the disrupted sequence RXKRK or RRKXK; substitution oftwo arginines results in the disrupted sequence XXKRK or XRKXK;substitution of three arginines results in the disrupted sequence XXKXK.Preferred is the substitution of all five basic amino acids resulting inthe sequence XXXXX, wherein X is a non-basic amino acid, preferably aneutral amino acid.

The modified huTSLP polypeptides of the invention also include aminoacid additions to the huTSLP amino acid sequence where one or more aminoacid residues are inserted into the furin cleavage sequence127-RRKRK-131, disrupting the RXXR pattern. For example, two or moreamino acids can be inserted, such as in the sequence 127-RRZ_(n)KRK-131where Z is not R or K, and n is not 1; one or more, and preferably twoor more amino acids can be inserted between arginines, or the sequence127-RZ_(n)RKRK-131, where Z is not R or K, and n is not 2; and the like.Preferably, n is 3, 4, or 5, and Z is a neutral amino acid.

Exemplary Modified Human TSLP Polypeptides

Exemplary Modified Human TSLP Polypeptides

X (*) can designate an amino acid substitution, deletion, insertion, orcombination of these that disrupts the activity of the furin cleavagesite.

In one exemplary embodiment, for example, set forth in SEQ ID NO:10, allof the amino acids designated by X are modified to be any amino acid,preferably a neutral amino acid, other than R or K. In anotherembodiment, one or more, and preferably two or more of X is an aminoacid deletion, most preferably two or more arginine (R) residues aredeleted, and most preferably each X represents a deleted amino acid. Inanother embodiment, one or more, and preferably two or more of X is anamino acid substitution that is not K or R, and is preferably neutralamino acid. In this embodiment, XXXXX can be, for example, XRXRX, XRXRK,RXRXX, or RXRXK.

As set forth in SEQ ID NO: 18, Z(**) can be any amino acid that is not Ror K, and preferably is a neutral amino acid. As discussed above, n canbe any number that disrupts the RXXR pattern, for example, n can be 1 orgreater, and preferably is 3, 4, or 5. Other exemplary methods fordeactivating the furan cleavage site pattern RXXR and particularlyRXR/KR will be apparent and are encompassed in the invention.

Examples of modified huTSLP polypeptides presented above includepolypeptides having the amino acid sequences set forth in SEQ ID NO: 10,12, 14, 16, 17, or 18, as well as polypeptides having an amino acidsequence which is substantially similar to these sequences, that is,having at least 80% identity to these amino acid sequences, andretaining resistance to furin cleavage as well as having at least oneTSLP activity. Human TSLP polypeptide activity can be readilydetermined, for example, by subjecting a variant, derivative, orfragment of a human TSLP polypeptide to the BAF/HRT bioassay describedin Example 3 below, or using the NAG8/7 cell proliferation assays asdescribed by Friend et al., supra, or to STAT5 activation assays asdescribed by Levin et al., supra.

The modified huTSLP polypeptides may be membrane bound or soluble,secreted polypeptides. In one embodiment, the soluble modifiedpolypeptide may include all or part of the extracellular domain, butlack the transmembrane region, which would cause retention of thepolypeptide on a cell membrane. Human TSLP polypeptides include variantsof the polypeptide encoded by SEQ ID NO:4 having at least 80% identityin amino acid sequence to SEQ ID NO:4 and retaining at least one TSLPfunction, as well as fragments thereof such as the soluble domainretaining a TSLP function.

Useful derivatives of the modified polypeptides of the inventioninclude, for example, modified human TSLP polypeptides attached to atleast one additional chemical moiety, or to at least one additionalheterologous polypeptide to form covalent or aggregate conjugate such asglycosyl groups, lipids, phosphate, acetyl groups, or C-terminal orN-terminal fusion proteins and the like. Preferred heterologouspolypeptides include those that facilitate purification, stability,cellular or tissue targeting, or secretion of the modified human TSLP,such as fusion proteins with the Fc polypeptide.

Modifications of the amino acid sequence of human TSLP polypeptides canbe accomplished by any of a number of known techniques. For example,mutations can be introduced at particular locations by known proceduressuch as oligonucleotide-directed mutagenesis (Walder et al., 1986, Gene,42:133; Bauer et al., 1985, Gene 37:73; Craik, 1985, BioTechniques,12-19; Smith et al., 1981, Genetic Engineering: Principles and Methods,Plenum Press; and U.S. Pat. No. 4,518,584 and U.S. Pat. No. 4,737,462).

The modified human TSLP polypeptides of the present invention arepreferably provided in an isolated form, and preferably aresubstantially purified. The polypeptides can be recovered and purifiedfrom recombinant cell cultures by known methods, including ammoniumsulfate or ethanol precipitation, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,and lectin chromatography. In a preferred embodiment, high performanceliquid chromatography (HPLC) is employed for purification.

Modified human TSLP can be fused to heterologous regions used tofacilitate purification of the polypeptide. Many of the availablepeptides (peptide tags) allow selective binding of the fusion protein toa binding partner. Non-limiting examples of peptide tags include 6-H is,thioredoxin, hemaglutinin, GST, and the OmpA signal sequence tag. Abinding partner that recognizes and binds to the peptide can be anymolecule or compound including metal ions (for example, metal affinitycolumns), antibodies, antibody fragments, and any protein or peptide,which binds the heterologous peptide to permit purification of thefusion protein.

Fragments spanning a modified furin cleavage site, including a fragmentwhere the furin cleavage site has been deleted, can be used to generatespecific antibodies against modified huTSLP polypeptides. The fragmentsshould be short, between 5 and 20 amino acids, and preferably between 5and 10 amino acids. Using known selection techniques, specific epitopescan be selected and used to generate monoclonal or polyclonalantibodies. Such antibodies have utility in the assaying proteaseresistant huTSLP activity, specifically identifying the expression ofprotease resistant huTSLP, and in the purification of the modifiedhuTSLP from cell culture.

Modified TSLP Polynucleotide Sequences

The invention also provides isolated nucleic acid molecules whichcomprise polynucleotides encoding the modified huTSLP polypeptides ofthe present invention. Polynucleotides of the invention include thosehaving an in-frame nucleotide sequence modification that disrupts orotherwise deactivates the codons that encode the furin cleavage siteRRKRK [SEQ ID NO: 6] positioned at approximately amino acid residues127-131 of SEQ ID NO: 4, such as, for example, the polynucleotidesequence AGG AGA AAA AGG AAA [SEQ ID NO: 5]. Suitable modificationsinclude in-frame nucleic acid substitutions, deletions, additions, orcombinations of these, that alter the sequence that encodes RRKRK todisrupt the encoded furin cleavage site pattern RXXR, particularlyRX(R/K)R. For example, in one embodiment the sequence: AGA AAA AGG AAAGTC [SEQ ID NO: 7] encoding an amino acid sequence RKRKV [SEQ ID NO: 8]is deleted.

Exemplary Human TSLP Mutant Polynucleotides

Exemplary Human TSLP Mutant Polynucleotides

x (*) can designate any in-frame nucleotide substitution, deletion,insertion, or combination of these, that disrupts the activity of thefuran cleavage site.

In one exemplary embodiment set forth in SEQ ID NO: 9, each xxx encodesany amino acid except for R or K, preferably a neutral amino acid. Inanother embodiment, one or more, and preferably two or more codons xxxare deleted, most preferably two or more codons encoding arginine (R)residues are deleted, and most preferably each xxx represents a deletedcodon. In another embodiment, one or more, and preferably two or more ofx are nucleotide substitutions that do not form codons encoding K or R,and preferably encode neutral amino acids. In a further embodiment, oneor more codons are inserted to disrupt the amino acid sequence of thefurin cleavage site, as discussed above, for example, RRKZ_(n)RK. Otherexemplary methods for modifying the codons to deactivate the furincleavage site pattern RXXR and particularly RXR/KR will be apparent andare encompassed in the invention.

Therefore, modified huTSLP polynucleotides of the invention includepolynucleotides having in-frame deletions, substitutions, or additionsto SEQ ID NO: 3, as long as the addition, deletion, or substitutiondeactivates the cleavage site and encodes a furin resistant huTSLPpolypeptide molecule which retains a TSLP activity. In addition, thepolynucleotides of the invention encompasses polynucleotides havingsequences which are substantially similar to this modified SEQ ID NO: 3,or a fragment of SEQ ID NO:3, and which encode modified TSLPpolypeptides which retain both at least one TSLP activity and furinresistance.

As used herein, a nucleic acid molecule is “substantially similar to”another nucleic acid molecule if its polynucleotide sequence is at least80% identical, preferably 90% identical, more preferably 95% identical,more preferably 98% identical, and most preferably 99% identical to thesequence of the second nucleic acid molecule, and if it encodes amodified TSLP polypeptide of the present invention retaining both a TSLPactivity and furin resistance. Polynucleotide sequence identity isdetermined by known methods, for example by aligning two sequences in asoftware program such as the MACAW program created by Greg Schuler. Inaddition, the percent identity may be determined by visual inspectionand mathematical calculation, or by comparing sequence information usingthe GAP computer program, version 6 described by Devereux et al. Nucl.Acids Res. 12:387 (1984), and available from the University of WisconsinGenetics Computer Group (UWGCG).

The modified huTSLP polynucleotides of the present invention can becDNA, chemically synthesized DNA, DNA amplified by PCR, RNA, orcombinations thereof. Due to the degeneracy of the genetic code, two DNAsequences can differ and yet encode identical amino acid sequences. Thepresent invention thus provides a nucleic acid molecule having apolynucleotide sequence encoding a modified huTSLP polypeptide. Thenucleic acid molecules of the present invention having a polynucleotidesequence encoding a polypeptide which is substantially similar to SEQ IDNO: 4 and modified to inactivate the furin cleavage site RRKRK. As usedherein, “substantially similar” refers to a polypeptide having at least80% identity in amino acid sequence to the modified SEQ ID NO: 4,Wherein the polypeptide retains both resistance for furin cleavage and aTSLP activity.

The present invention also includes polynucleotides having SEQ ID NO: 9,11, 13, or 15 and polynucleotides which are substantially similar tothese polynucleotide sequences. In addition, the present inventionprovides polynucleotides encoding the polypeptides of SEQ ID NO: 10, 12,14, 16, 17, or 18, and polynucleotides encoding polypeptides which aresubstantially similar to these polypeptides.

Useful fragments of the polynucleotides of the invention include probesand primers. These can be used, for example, in PCR methods to amplifyand detect the presence of modified huTSLP polynucleotides in vitro, aswell as in Southern and Northern blots for analysis of proteaseresistant huTSLP. Cells transiently or stably overexpressing theprotease resistant huTSLP polynucleotide molecules of the invention canalso be identified by the use of such probes. Methods for the productionand use of such primers and probes are known.

Other useful fragments include antisense or sense oligonucleotidescomprising a single-stranded nucleic acid sequence capable of binding toa target modified huTSLP mRNA (using a sense strand) or DNA (using anantisense strand) sequence.

Vectors and Host Cells

The present invention provides vectors containing the polynucleotidesdescribed above, as well as host cells transformed with such vectors.Any of the polynucleotides molecules of the invention can be containedin a vector, which generally includes a selectable marker and an originof replication, for propagation in a host. The vectors further includesuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect genes,operably linked to the modified huTSLP polynucleotide molecule. Examplesof such regulatory sequences include transcriptional promoters,operators, or enhancers, mRNA ribosomal binding sites, and appropriatesequences that control transcription and translation. Nucleotidesequences are operably linked when the regulatory sequence functionallyrelates to the DNA encoding the target protein. Thus, a promoternucleotide sequence is operably linked to a modified huTSLP DNA sequenceif the promoter nucleotide sequence directs the transcription of themodified TSLP sequence.

Selection of suitable vectors for the cloning of protease resistanthuTSLP polynucleotide molecules of this invention will depend upon thehost cell in which the vector will be transformed, and, whereapplicable, the host cell from which the target polypeptide is to beexpressed. Suitable host cells include prokaryotes, yeast, and highereukaryotic cells, each of which is discussed below. Modified huTSLPpolypeptides are often expressed in mammalian cells.

The modified huSLP polypeptides to be expressed in host cells can alsobe a fusion proteins comprising the TSLP polypeptide and at least oneheterologous polypeptide. As discussed above, heterologous polypeptidescan be fused to the TSLP polypeptide to facilitate, for example,secretion, stability, purification, and/or targeting of the modifiedhuTSLP polypeptide. Examples of fusions proteins provided by the presentinvention includes fusions of modified TSLP polypeptides with, forexample Fc polypeptides and leucine zipper domains to promote theoligomerization of the TSLP polypeptides as described in WO 00/29581.

In another embodiment, a nucleotide sequence encoding an appropriatesignal peptide can be incorporated into an expression vector. A nucleicacid sequence encoding a signal peptide (secretory leader) can be fusedin-frame to the modified huTSLP sequence so that modified huTSLP istranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cell promotesextracellular secretion of the polypeptide. Preferably, the signalsequence will be cleaved from the modified huTSLP polypeptide uponsecretion of the polypeptide from the cell. Non-limiting examples ofsignal sequences that can be used in practicing the invention includethe yeast I-factor and the honeybee melatin leader in Sf9 insect cells.

Suitable host cells for expression of target polypeptides of theinvention include prokaryotes, yeast, and higher eukaryotic cells; mostpreferred are mammalian cells. Suitable prokaryotic hosts that can beused for the expression of these polypeptides include bacteria of thegenera Escherichia, Bacillus, and Salmonella, as well as members of thegenera Pseudomonas, Streptomyces, and Staphylococcus. For expression inprokaryotic cells, for example E. coli, the polynucleotide moleculeencoding the modified huTSLP polypeptide preferably includes anN-terminal methionine residue to facilitate expression of therecombinant polypeptide. The N-terminal Met can optionally be cleavedfrom the expressed polypeptide.

Expression vectors for use in prokaryotic hosts generally comprise oneor more phenotypic selectable marker gene. Such gene generally encodes,for example, a protein that confers antibiotic resistance or thatsupplies an auxotrophic requirement. A wide variety of such vectors arereadily available from commercial sources. Examples include pSPORTvectors, pGEM vectors (Promega), pPROEX vectors (LTI, Bethesda, Md.),Bluescript vectors (Stratagene), and pQE vectors (Qiagen).

Modified huTSLP can also be expressed in yeast host cells from generaincluding Saccharomyces, Pichia, and Kluveromyces. Preferred yeast hostsare S. cerevisiae, and P. pastoris. Yeast vectors will often contain anorigin of replication sequence from a 2T yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Vectors replicable in both yeast and E. coli(termed shuttle vectors) can also be used. In addition to theabove-mentioned features of yeast vectors, a shuttle vector will alsoinclude sequences for replication and selection in E. coli. Directsecretion of the target polypeptides expressed in yeast hosts can beaccomplished by the inclusion of nucleotide sequence encoding the yeastI-factor leader sequence at the 5′ end of the modified huTSLP encodingnucleotide sequence.

Insect host cell culture systems can also be used for the expression ofthe modified huTSLP polypeptides. The target polypeptides of theinvention are preferably expressed using a baculovirus expressionsystem, as described, in a review by Luckow and Summers, 1988,Bio/Technology 6:47.

In the preferred embodiment, the modified huTSLP polypeptides of theinvention are expressed in mammalian host cells. Non-limiting examplesof suitable mammalian cell lines include the COS-7 line of monkey kidneycells (Gluzman et al., 1981, Cell 23:175), Chinese hamster ovary (CHO)cells (Puck et al., 1958, Proc. Natl. Acad. Sci. USA, 60:1275-1281; CV-1cells (ATC CRL-10478); 293 cells, COS cells, and human cervicalcarcinoma cells (HELA) (ATCC CCL 2).

The choice of a suitable expression vector for expression of the targetpolypeptides of the invention will depend upon the specific mammalianhost cell to be used. Examples of suitable expression vectors includepDC 409 (McMahan et al., 1991, EMBO J. 10:2821), pDC 317 (Source),pcDNA3.1/Hygro (Invitrogen), pSVL (Pharmacia Biotech) and the vectorsdescribed in WO 01/27299.

Expression vectors for use in mammalian host cells can includetranscriptional and translational control sequences derived from viralgenomes. Commonly used promoter sequences and enhancer sequences thatcan be used to express the modified human TSLP include, but are notlimited to, those derived from human cytomegalovirus (CMV), Adenovirus2, Polyoma virus, and Simian virus 40 (SV40). Methods for theconstruction of mammalian expression vectors are disclosed, for example,in Okayama and Berg, 1983, Mol. Cell. Biol. 3:280; Cosman et al., 1986,Mol. Immunzol. 23:935; Cosman et al., 1984, Nature 312:768;EP-A-0367566; and WO 91/18982.

Modification of a protease resistant huTSLP polynucleotide molecule tofacilitate insertion into a particular vector (for example, by modifyingrestriction sites), ease of use in a particular expression system orhost (for example, using preferred host codons), and the like, are knownand are contemplated for use in the invention. Genetic engineeringmethods for the production of modified human TSLP polypeptides includethe expression of the polynucleotide molecules in cell free expressionsystems, in cellular hosts, in tissues, and in animal models, accordingto known methods.

Compositions

The invention provides compositions containing a substantially purifiedmodified huTSLP polypeptide of the invention and a carrier. Fortherapeutic applications, the invention provides compositions adaptedfor pharmaceutical use, for example, containing a pharmaceuticallyacceptable carrier. Pharmaceutical compositions of the invention areadministered to cells, tissues, or patients, for example, to induce theactivity of B and T cells; and for therapeutic treatment, for example,in stimulating immune cell proliferation and development inimmuno-suppressed patients, for example AIDS. The pharmaceuticalcompositions containing a modified huTSLP polypeptide are also useful asvaccine adjuvants, for example, useful for obtaining long-term immunity.

The invention also provides reagents, compositions, and methods that areuseful for analysis of B and T cell activity; for analysis of STAT5activity; and for analysis of the inhibitory/stimulatory effects ofsignal molecules involved in innate immune system responses.

Antibodies

The polypeptides of the present invention, in whole or in part, can beused to generate antibodies that are useful in assays for detectingmodified huTSLP polypeptide expression and for purification ofoverexpressed modified human TSLP. Antibodies against modified TSLPpolypeptides can be used as an antagonist to TSLP activity in a system.Methods for the selection of peptide epitopes and production ofantibodies are known. See, for example, Antibodies: A Laboratory Manual,Harlow and Land (eds.), 1988 Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Monoclonal Antibodies, Hybridomas: A New Dimensionin Biological Analyses, Kennet et al. (eds.), 1980, Plenum Press, NewYork.

In addition to the production of antibodies, all or a portion of themodified TSLP polypeptide of the invention can be used, for example, asa targeting moiety to target binding to cells and tissues expressingTSLP receptors.

Assays

Human TSLP activity can be identified and measured using a number ofassays including assays involving huTSLP effects on B and T cellproliferation and development. One such assay is described in Example 3.BAF cells expressing human TSLP receptors (BAF/HTR) which require activeTSLP for proliferation can be used to measure TSLP activity as describedin Example 3 herein. Additional assays for hTSLP activity include, forexample, an assay measuring induction of T cell growth from human bonemarrow by TSLP is described in WO 00/29581. Another TSLP activity is theability to activate STAT5 as described in the reference to Levin et al.,1999, J. Immunol. 162:677-683, and in Example 4 herein.

These assays can be used to determine and quantitate on a relative basisTSLP activity, for various modified TSLP polypeptides including variantsand derivates. In addition, these assays can be used identify agentswhich act to modify TSLP activity, or eliminate TSLP activity. Forexample, a lower modified huTSLP activated test activity in the presenceof the test agent, compared with the absence of the test agent,indicates that the test agent has decreased the activity of the modifiedhuTSLP. A higher protease resistant huTSLP activated test activity inthe presence of the test agent than in the absence of the test agentindicates that the test agent has increased the activity of the proteaseresistant huTSLP. Stimulators and inhibitors of modified huTSLP can beused to augment, inhibit, or modify huTSLP mediated activity, andtherefore can have therapeutic uses. For example, inhibitors of modifiedhuTSLP can be useful to reduce B and T cell activity, for example inautoimmune diseases or in patients undergoing organ transplants.

Therapeutic Applications

The modified huTSLP polypeptides of the invention can be usedtherapeutically in the same manner known for the therapeutic use of thehuTSLP polypeptide, as discussed in the publications referenced above.huTSLP is effective to stimulate B and T cell activities. For example,huTSLP, and preferably micromolar amounts of soluble modified huTSLPinduces B and T cell differentiation, proliferation, and activation.Such administration is therapeutically useful in the treatment ofbacterial and viral infections, as well as in the treatment of tumorcells and autoimmune deficiencies.

Further, the polypeptides of the present invention can be used alone orin combination with IL-7 to reconstitute a patient's immune systemfollowing autologous bone marrow transplantation (see for exampleAbdul-Hai et al., 1996, Experimental Hematology, 24:1416-1422). TSLP,due to its known effects on STAT5, can also be used in therapiestargeted to modify STAT5 effects on a patient (see Richer et al., 1998,J. Biol. Chem., 273(47):31317-31326; Davey et al., 1999, Am. J. Hum.Genet., 65:959-965; Nosaka et al., 1999, EMBO J, 18(17):4754-4765).

Modified human TSLP polynucleotides and polypeptides, including vectorsexpressing modified huTSLP, of the invention can be formulated aspharmaceutical compositions and administered to a host, preferablymammalian host, including a human patient, in a variety of forms adaptedto the chosen route of administration. The compounds are preferablyadministered in combination with a pharmaceutically acceptable carrier,and can be combined with or conjugated to specific delivery agents,including targeting antibodies and/or cytokines.

Modified human TSLP can be administered by known techniques, such asorally, parentally (including subcutaneous injection, intravenous,intramuscular, intrasternal or infusion techniques), by inhalationspray, topically, by absorption through a mucous membrane, or rectally,in dosage unit formulations containing conventional non-toxicpharmaceutically acceptable carriers, adjuvants or vehicles.Pharmaceutical compositions of the invention can be in the form ofsuspensions or tablets suitable for oral administration, nasal sprays,creams, sterile injectable preparations, such as sterile injectableaqueous or oleagenous suspensions or suppositories.

For oral administration as a suspension, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can contain microcrystalline cellulose forimparting bulk, alginic acid or sodium alginate as a suspending agent,methylcellulose as a viscosity enhancer, and sweeteners or flavoringagents. As immediate release tablets, the compositions can containmicrocrystalline cellulose, starch, magnesium stearate and lactose orother excipients, binders, extenders, disintegrants, diluents andlubricants known in the art.

For administration by inhalation or aerosol, the compositions can beprepared according to techniques well-known in the art of pharmaceuticalformulation. The compositions can be prepared as solutions in saline,using benzyl alcohol or other suitable preservatives, absorptionpromoters to enhance bioavailability, fluorocarbons or othersolubilizing or dispersing agents known in the art.

For administration as injectable solutions or suspensions, thecompositions can be formulated according to techniques well-known in theart, using suitable dispersing or wetting and suspending agents, such assterile oils, including synthetic mono- or diglycerides, and fattyacids, including oleic acid.

For rectal administration as suppositories, the compositions can beprepared by mixing with a suitable non-irritating excipient, such ascocoa butter, synthetic glyceride esters or polyethylene glycols, whichare solid at ambient temperatures, but liquefy or dissolve in the rectalcavity to release the drug.

Preferred administration routes include orally, parenterally, as well asintravenous, intramuscular or subcutaneous routes. More preferably, thecompounds of the present invention are administered parenterally, i.e.,intravenously or intraperitoneally, by infusion or injection. In oneembodiment of the invention, the compounds can be administered directlyto a tumor by tumor injection; or by systemic delivery by intravenousinjection.

Solutions or suspensions of the compounds can be prepared in water,isotonic saline (PBS) and optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethylene,glycols, DNA, vegetable oils, triacetin and mixtures thereof. Underordinary conditions of storage and use, these preparations can contain apreservative to prevent the growth of microorganisms.

The pharmaceutical dosage form suitable for injection or infusion usecan include sterile, aqueous solutions or dispersions or sterile powderscomprising an active ingredient which are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions.In all cases, the ultimate dosage form should be sterile, fluid andstable under the conditions of manufacture and storage. The liquidcarrier or vehicle can be a solvent or liquid dispersion mediumcomprising, for example, water, ethanol, a polyol such as glycerol,propylene glycol, or liquid polyethylene glycols and the like, vegetableoils, nontoxic glyceryl esters, and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the formation ofliposomes, by the maintenance of the required particle size, in the caseof dispersion, or by the use of nontoxic surfactants. The prevention ofthe action of microorganisms can be accomplished by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, buffers, or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the inclusion in thecomposition of agents delaying absorption, for example, aluminummonosterate hydrogels and gelatin.

Sterile injectable solutions are prepared by incorporating the compoundsin the required amount in the appropriate solvent with various otheringredients as enumerated above and, as required, followed by filtersterilization. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying techniques, which yield a powder of theactive ingredient plus any additional desired ingredient present in thepreviously sterile-filtered solutions.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Recognition and Modification of Furin Cleavage Site

The nucleic acid sequences encoding murine TSLP (GenBank accessionnumber AF232937) [SEQ ID NO: 3] and human TSLP [SEQ ID NO: 5] weredisclosed in PCT application WO 00/29581. Production of usefulquantities of human TSLP cDNA in mammalian cells is hampered, however,as expression in mammalian cells often yields a degraded product.

Expression of human recombinant TSLP in mammalian cells providedsubstantially lower quantities of full length recombinant protein thanexpected. A portion the expressed protein was in a cleaved, fragmentedform, having a major degradation product of 6 kD. In contrast to humanTSLP, murine TSLP was not degraded when expressed in mammalian cells.The nucleic acid and amino acid sequences of the human and murine TSLPwere then compared. As shown in Table 1, comparison of the human TSLPamino acid sequence with a murine TSLP amino acid sequence revealed aseries of residues, beginning at residue 128, found exclusively in thehuman TSLP (128-RKRKV-132). Upon further investigation, it wasdetermined that the residues represented a furin cleavage site(127-RRKRK-131). Importantly, the position of the furin cleavage sitecorrelated with release of an approximate 6 kD C-terminal fragment ofhuman TSLP.

The huTSLP amino acid sequence includes an N-terminal hydrophobic regionthat functions as a signal peptide followed by a series of 4 helixesforming a four-helix bundle cytokine structure. The furin cleavage siteis positioned about 8 amino acids before the start of the fourth helixof the four-helix bundle. Truncation of the protein at the cleavage sitecan result in an inactivated human TSLP protein. TABLE 1 Comparison ofmurine and human TSLP polypeptides

When TSLP protein is expressed and isolated from mammalian cellcultures, and analyzed, for example, by electrophoresis, a number ofpolypeptides result, shown as numerous bands on a gel. The mostprominent band in the mixture of proteins has a molecular weight ofapproximately 6 kD. The amino acid sequence of the 6 kD fragmentcorresponds to the C-terminal end of TSLP, suggesting a cleavage pointat the furin cleavage site, RRKRK. This data provides direct evidencethat degradation of human TSLP expressed in mammalian cells results fromcleavage at the furin cleavage site.

Example 2 Mutagenesis of Furin Site in Human TSLP

Site directed mutagenesis was used to inactivate the furin cleavage sitefrom the human TSLP poly-H is FLAG transcript, using 313-human-TSLPHis-FLAG (#14095) (1 mg/ml) as the template. A series of polymerasechain reaction (PCR) reactions was used to delete the nucleotidesequence: AGA AAA AGG AAA GTC [SEQ ID NO: 7] that encodes the huTSLPsegment containing the furin cleavage site: RKRKV [SEQ ID NO: 8]. Inaddition, a combination of primers was designed to create a Sal-1restriction site at the 5′ end and a Not-1 site at the 3′ end. Theprimers were as follows: 1. Forward (Sal-1): 5′-GTCGACGCCACCATGTTCCCT-3′[SEQ ID NO:19] 2. Forward: 5-′ATGAAGAAGAGGACAACCAATAAATGTC-3′ [SEQ IDNO:20] 3. Reverse: 5′-GACATTTATTGGTTGTCCTCTTCTTCAT-3′ [SEQ ID NO:21] 4.Reverse (Not-1): 5′-AGCGGCCGCTCATTTGTCGTC-3′ [SEQ ID NO:22]

The polynucleotide sequence of human TSLP is shown below:

HUMAN TSLP (GenBank AY037115)

HUMAN TSLP (GenBank AY037115)

A PCR product of 409 bases was formed using primers 1 and 3 in a firstPCR reaction. Primer 1 includes a Sal-1 restriction site, while primer 3deletes the 15 base furin cleavage sequence. A second PCR product of 162bases was formed using primers 2 and 4 in a second PCR reaction, withprimer 4 includes a NOT-1 restriction site, while primer 2 deletes the15 base furin cleavage site.

The PCR reactions contained 10 Tl of Amplitaq 10× buffer; 1Tl Amplitaq;2Tl dNTPs (10 pM each); 40 pM each primer; 1Tl template; water to afinal volume of 100Tl. The PCR was performed on a Perkin Elmer-Gene AmpPCR Systems 2400 machine, at: 1 cycle of 94° C., 2:00 minutes; 30 cyclesof 94° C., 0:30 minutes, 50° C., 0:15 minutes, and 72° C., 1:00 minute;and one cycle of 72° C., 2:00 minutes.

PCR products were purified in 1% low melt agarose gels. Appropriatesized-bands were excised from the gel and the DNA was purified using aHigh Pure PCR Product Purification Kit obtained from BoehringerMannheim. The gel-purified 409 and 162 base products were combined withprimers 1 and 4 to produce a 558 base pair PCR product that containedthe full length human TSLP polyHis-FLAG sequence lacking the 15 basepair region encoding the furin cleavage site.

The reaction solution contained: 10Tl Amplitaq 10× buffer; 1Tl Amplitaq;2Tl (10 pM each) dNTP; 15.4Tl (40 pM) Primer 1; 16 Tl (40 pM) Primer 4;1Tl (41 ng) PCR Product A; 2Tl (119 ng) PCR Product B; and water to afinal volume of 100 Tl. As above, the PCR reaction/conditions werecarried out in the Perkin Elmer-Gene Amp PCR Systems 2400 machine. Atthe end of the reaction, the 558 base pair product was separated on a 1%low-melt agarose gel and purified.

Purified modified human TSLP sequence was ligated into vector pGEM-T(Promega), using the reagents supplied with the vector kit: 1Tl pGEM-Tvector; 5Tl 2× ligation buffer; 1Tl ligase; and 1Tl 558 base pairmodified human TSLP (12 ng). The reaction solution was left at roomtemperature for one hour. The ligation mixture (2Tl) was combined with40 Tl of DH10I-electrocompetent E. coli, and electroporated into thebacteria. The electroporated bacteria were then transferred to 0.9 ml ofSOC solution and shaken for one hour at 37° C.

A volume of 0.1Tl of this solution was spread on ampicillin-resistantplates and incubated at 37° C. overnight. Colonies were picked andinoculated into 4 mL of LB broth containing ampicillin. After overnightincubation on a shake platform at 37° C., the plasmid DNA was purifiedand digested with NOT-1/Sal-1 to confirm the correct size of the insert.The pGem-T vector with the 558 base pair insert was sequenced to confirmthat the molecular manipulations had produced the desired mutation.

pGEM-T vector was digested with Not-I and Sal-1, and the 558 base pairinsert subcloned into expression vectors pDC 409 and pDC317. Digestionand ligation reactions were performed as is well known in the art.Expression vectors were then used to produce either transientlytransfected CV-1 cells (ATCC CRL-10478) or to make stably expressing CHOcells. Note that for comparison, a control expression vector encodinghuman TSLP having an intact furin cleavage site was used to produce bothtransient and stable transfected cells.

HuTSLP and modified huTSLP protein were each expressed in CV-1 cells asa HIS, Flag fusion protein. The expressed protein was purified usingIMAC (immobilized metal affinity chromatography, using themanufacturer's instructions (Qiagen)). Analysis of the expressed proteinon SDS-PAGE under reducing and non-reducing conditions demonstrated theproduction of modified huTSLP.

The constructed, modified human TSLP sequence, having the furin cleavagesite removed, was expressed as full-length human TSLP protein inmammalian culture (CV-1 cells). When compared to the non-modified humanTSLP, little or no degradation product was produced with expression ofthe furin-site deleted TSLP, demonstrating that the furin site was, infact, the site responsible for the fragmentation of recombinant humanTSLP.

Example 3 Active Modified Human TSLP

The activity of the modified huTSLP, produced as described for Example2, was verified using a BAF/HRT cell bioassay. The BAF/HTR bioassayutilizes a murine pro B lymphocyte cell line, which has been transfectedwith the human TSLP receptor (cell line obtained from Steven F. Ziegler,Virginia Mason Research Center, Seattle, Wash.). The TSLPR DNA sequencewas deposited with Genbank, (accession number AF201963) and is describedin Pandey et al., 2000, Nat Immun 1(1), 59-64. These cells are dependentupon huTSLP for growth, and proliferate in a dose-dependent manner inresponse to active huTSLP added in test samples.

Titrations of samples and standards were performed in a 96-wellmicrotiter format. A baseline quantity of BAF/HRT cells were added toeach well. Samples of modified huTSLP and standards were added to thewells. Following an incubation period, cell proliferation was measuredby the addition of Alamar Blue dye I (Biosource International Catalog#DAL1100, 10 uL/well). Metabolically active BAF/HRT cells take up andreduce Alamar Blue, which leads to change in the fluorescent propertiesof the dye. The number of fluorescent units produced in this assay bythe modified, protease resistant huTSLP was similar to that of thereference unmodified huTSLP, showing that the modified huTSLP wasequally active to unmodified huTSLP.

Example 4 Modified huTSLP Activates STAT5

The ability of modified huTSLP of the invention to activate STAT5 isanalyzed according to the method described in Levin et al., 1999 supra.Briefly, NAG8/7 cells are cytokine starved for 4-5 hours, thenstimulated at 10⁷ cells/ml with 100 ng/ml modified human TSLP.Unmodified huTSLP is used as a control. Post incubation, cells areharvested, washed, and lysed. Stimulated cell lysates are analyzed byimmunoblot assay, and demonstrate modified huTSLP activity when comparedwith control.

The invention is described herein with reference to specific examples.Various changes and modifications can be made to these examples that arewell within the scope of the invention. Numerous other changes can bemade that are readily suggested to those skilled in the art and that areencompassed in the spirit of the invention disclosed herein and asdefined in the appended claims.

All publications cited herein are hereby incorporated by reference.

1-14. (canceled)
 15. An isolated protein comprising a modified humanthymic stromal lymphopoietin (TSLP) polypeptide, wherein the polypeptideis selected from the amino acid sequences consisting of SEQ ID NO: 10,12, 14, 16, 17 and
 18. 16. (canceled)
 17. The protein of claim 15wherein the polypeptide is fused to a heterologous protein.
 18. Theprotein of claim 17, wherein the heterologous protein is acell-targeting moiety.
 19. The protein of claim 18, wherein thecell-targeting moiety is an antibody that binds a cell surface antigen.20. The protein of claim 18, wherein the cell-targeting moiety is aligand that binds a cell surface receptor.
 21. The protein of claim 17,wherein the heterologous protein is a peptide tag.
 22. The protein ofclaim 17, wherein the heterologous protein is an Fc polypeptide. 23-42.(canceled)